Despite the world's reliance on natural products from plants and microbes to treat and cure serious diseases, many fundamental questions remain to be answered as to how and why these medicines are produced in nature. Knowing more about the metabolism of these compounds will lead to simpler and more rational strategies for strain improvement. Strain improvement speeds up the drug development process and helps to reduce the cost of new drugs (Mateles, 2000).
An enormous array of medically important chemical structures are made in nature, particularly by plants and microbes. These structures fall into chemical classes based on shared routes of biosynthesis. One well-studied class of compounds is the polyketides, perhaps best characterized by the macrolide antibiotics, of which erythromycin is a primary example. Erythromycin and its derivatives, marketed under trade names such Biaxin®, Rulid®, and Zithromax®, are in wide use in the world today. Erythromycin's biosynthesis has been studied for over 50 years and so it is a widely used model system for secondary metabolite production.
Like many secondary metabolites, erythromycin is a tailored polymer. The building blocks are one molecule of propionic acid and 6 molecules of methylmalonic acid in their CoA forms (Omura, 1984). Tailoring steps include the addition of two sugars, the addition of a methyl group to one sugar, and the addition of two hydroxyl groups to the polyketide polymer backbone. Despite agreement on the identity of the chemical building blocks, scientists are still unsure of the source of the propionic acid and methylmalonic acid that are used to form the molecule. Knowing this key piece of information would help lead the way to development of genetic and process manipulations in order to boost production of the antibiotic.
Originally it was reported that succinyl-CoA is the major source of methylmalonyl-CoA via the enzyme methylmalonyl-CoA mutase (MCM) (Hunaiti and Kolattukudy, 1984). Propionyl-CoA was reported to come from decarboxylation of methylmalonyl-CoA (Hsieh and Kolattukudy, 1994). These early results implied that precursors for erythromycin biosynthesis are taken at the expense of central metabolism in a reverse-anaplerotic reaction. Consistent with these results, in a different macrolide producing host, when the mutAB genes, coding for MCM, were overexpressed, a macrolide antibiotic was overproduced (Zhang et al., 1999).
Amino acid catabolism has also been identified as an important source of precursors for macrolide biosynthesis (Omura et al., 1983, 1984; Dotzlaf et al., 1984). When branched chain amino acids such as valine, −isoleucine, leucine, or valine catabolites (propionate and isobutyrate) and threonine were added to the fermentation medium they boosted production of a macrolide antibiotic and its polyketide derived precursors (Omura et al., 1983, 1984, Tang et al., 1994). Conversely, when valine catabolism was blocked at the first step, (valine dehydrogenase, vdh), production of two different macrolide antibiotics went down 4-to-6-fold (Tang et al., 1994). These results pointed to amino acid catabolism, in particular branched-chain amino acid (BCAA) catabolism, as another vital source of macrolide antibiotic precursors in actinomycetes.
Surprisingly, when the branched-chain amino acid catabolic pathway was blocked at a later step in propionyl-CoA carboxylase, it did not lead to a reduction in macrolide production (Donadio et al, 1996). These results conflict with those of Dotzlaf et al, (1984), but they were obtained in a different macrolide-producing host and precursor feeding pathways have not yet been shown to operate universally in different hosts. Other workers also reported on this propionyl-CoA carboxylase reaction (Hunaiti and Kolatukuddy, 1982). Hsieh and Kolattukudy, 1994 cloned a gene that recent BLASTX analyses now shows could not code for a carboxylase, and may have been cloned by mistake.
Methylmalonyl-CoA mutase, coded for by the mutAB gene pair, was originally cited by Hunaiti and Kolattukudy (1984) to be the key enzyme to provide methylmalonyl-CoA for erythromycin biosynthesis.
According to the conclusions of Hunaiti and Kolattukudy (1984) and Zhang et al., (1999) whose results indicated the source of methylmalonyl-CoA to be from succinyl-CoA, one would predict that a block in mutB should reduce or block production of the erythromycin. This direction for methylmalonyl-CoA mutase, though, is often referred to as the “reverse” direction, because the forward—or anaplerotic—direction towards succinyl-CoA is favored enzymatically by a factor of twenty to one (Kellermeyer, et al., 1964; Vlasie and Banerjee, 2003).